View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Developmental Biology 298 (2006) 234–247 www.elsevier.com/locate/ydbio

Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes ⁎ Chisa Shukunami , Aki Takimoto, Miwa Oro, Yuji Hiraki

Department of , Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan Received for publication 22 February 2006; revised 19 June 2006; accepted 22 June 2006 Available online 27 June 2006

Abstract

Tenomodulin (TeM) is a type II transmembrane glycoprotein containing a C-terminal anti-angiogenic domain and is predominantly expressed in tendons and ligaments. Here we report that TeM expression is closely associated with the appearance of tenocytes during chick development and is positively regulated by Scleraxis (Scx). At stage 23, when Scx expression in the syndetome has extended to the tail region, TeM was detectable in the anterior eight . At stage 25, TeM and Scx were both detectable in the regions adjacent to the myotome. Double positive domains for these genes were flanked by a dorsal TeM single positive and a ventral Scx single positive domain. At stage 28, the expression profile of TeM in the axial tendons displayed more distinct morphological features at different levels of the vertebrae. At stage 32 and later, Scx and TeM showed similar expression profiles in developing tendons. Retroviral expression of Scx resulted in the significant upregulation of TeM in cultured tenocytes, but not in chondrocytes. In addition, the misexpression of RCAS-cScx by electroporation into the hindlimb could not induce the generation of additional tendons, but did result in the upregulation of TeM expression in the tendons at stage 33 and later. These findings suggest that TeM is a late marker of tendon formation and that Scx positively regulates TeM expression in a tendon cell lineage-dependent manner. © 2006 Elsevier Inc. All rights reserved.

Keywords: Tenomodulin; Scleraxis; Tendon; Ligament; Tenocytes; Chick

Introduction them to each other (Kardon, 1998). In the developing tendons, tenocytes align in parallel arrays along the tendon axis and deposit The tendons are tough bands of dense fibrous connective tissue large amounts of extracellular matrices including collagens, that connect to the skeletal elements. Their high elastin and small leucin-rich proteoglycans (Kannus, 2000). tensile strength principally depends on dense regular bundles of Collagen fibers, mainly consisting of type I collagen, show a type I collagen fibers that are produced by elongated fibroblasts highly organized parallel structure around the tenocytes. A recent known as tenocytes. Tenocytes are aligned longitudinally between 3-D serial section reconstruction study has revealed that the the collagen fibers along the tendon axis (Benjamin and Ralphs, architecture characterized by parallel alignment of collagen fibers 1997; Kannus, 2000) and have the potential to communicate with in tendons is achieved through the fibripositor formation of one another via specific cellular processes and gap junctions that tenocytes during embryonic development (Canty and Kadler, could form the basis of a load sensing system, thereby allowing 2005; Canty et al., 2004). the tenocytes to modulate their extracellular matrices (Canty and At present, few genes have been reported as general tendon Kadler, 2005; McNeilly et al., 1996). markers that can be used to delineate the sequence of tendon During the early stages of musculoskeletal development, development (Edom-Vovard and Duprez, 2004). Despite their tendon progenitors proliferate and migrate to form a tendon unique architecture, most structural components of tendons are primordium. This tendon primordium then segregates into also expressed in other connective tissues. Among these, te- individual anatomically distinct tendons, which interact with nascin (TN) has been employed as a tendon marker, although it developing muscle masses and skeletal elements and connect is also expressed in cartilage and nerve (Kardon, 1998; Ros et al., 1995; Tucker et al., 1994). Recently, Scx has been reported ⁎ Corresponding author. Fax: +81 75 751 4633. as a marker of the progenitor populations of tendons and E-mail address: [email protected] (C. Shukunami). tenocytes (Cserjesi et al., 1995; Schweitzer et al., 2001). Based

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.06.036 C. Shukunami et al. / 298 (2006) 234–247 235 on the expression pattern of Scx, the syndetome containing the that Scx positively regulates the expression of TeM in a tendon tendon progenitor population was defined as the fourth lineage-dependent manner. compartment of the somites, in addition to myotome, sclerotome and dermomyotome (Brent et al., 2003). Ectopic application of FGF8 or FGF4 has been shown to induce the Materials and methods expansion of Scx expression in the sclerotomal cells near the Cloning of chick TeM myotome (Brent et al., 2003). The classical ERK MAP kinase pathway plays an important role in the regulation of Scx Chick EST cDNA clones (BM491558 and BM487464) showing homology expression by FGF signaling (Smith et al., 2005). The with human TeM were identified in the EST database of the National Center for restriction of Scx expression in the syndetome is regulated by Biotechnology Information (National Institutes of Health). A cDNA fragment of the Ets transcription factors Pea3 and Erm (Brent and Tabin, 551 bp was amplified from the cDNA prepared from the leg tendons of chick embryos at stage 43 by reverse transcription PCR. The following primers were 2004). In limb tendons, FGF4 also positively regulates Scx and designed to amplify the partial cDNA fragment, based on the nucleotide TN expression (Edom-Vovard et al., 2002). In contrast, Shh and sequences from the EST clones: forward (5′-CGAGCGCTCGTCCTCGCGCA- Pax-1 inhibit syndetome formation (Brentetal.,2003). CAGG-3′) and reverse (5′-GGAACTCCTTGTTCTGAATGG-3′). For the Compared with the upstream regulation of Scx expression, isolation of chick TeM cDNA clones, encompassing the entire coding region, λ little is known about the downstream events that are closely a gt11 cDNA library constructed from 10 day whole chicken embryos (Clontech) was employed. The inserts isolated from the phage clones were then associated with tenocyte differentiation. Moreover, to distin- subcloned into the EcoRI site of pBluescript SK (+) (Stratagene). Both cDNA guish tenocytes from tendon progenitors during tendon strands were sequenced using standard sequence primers for this vector and development, an additional tendon marker that can selectively gene-specific primers with a BigDye terminator v1.1 Cycle Sequencing Kit and identify a differentiated population of tendon fibroblasts is an ABI 310 Genetic Analyzer (Applied Biosystems). The obtained sequences required. were compiled and analyzed by Macvector (Accelrys Software Inc.). TeM is a type II transmembrane that is specifically In situ hybridization expressed in dense connective tissues including tendons, ligaments, the epimysium of skeletal muscle, the cornea and Chick embryos at different stages of development were obtained by incubation the sclera (Brandau et al., 2001; Oshima et al., 2003; Pisani et of fertilized white leghorn eggs at 38°C and staged according to the method al., 2004; Shukunami et al., 2001; Yamana et al., 2001). Mice described by Hamburger and Hamilton (Hamburger and Hamilton, 1992). In some lacking TeM display a severe decrease in tenocyte proliferation embryos, india ink was injected into the vitelline vein to visualize blood vessels. in newborn tendons and a disrupted adult collagen fibril The antisense and sense RNA probes for each gene under analysis were transcribed from expression plasmids with a digoxygenin (DIG) RNA labeling kit (Roche). structure (Docheva et al., 2005). The soluble form of TeM, For whole mount in situ hybridization, chick embryos were fixed in 4% containing the C-terminal ChM-I like domain, inhibits the paraformaldehyde (PFA), resolved in phosphate-buffered saline (PBS) overnight proliferation, migration and tube formation of vascular and gradually transferred into methanol by exchange of the PFA against increasing endothelial cells and also blocks the growth of malignant concentrations of methanol/PBS. After rehydration from the methanol, the melanoma by inhibiting angiogenesis (Oshima et al., 2004; embryos were hybridized with DIG-labeled antisense RNA probes, and/or DIG- labeled and fluorescein labeled antisense RNA probes. Following hybridization at Shukunami et al., 2005). Thus, TeM may prove to be a 65°C overnight, the DIG-labeled molecules were detected using NBT/BCIP promising phenotypic marker of the later events during tendon (Roche) as the substrate for the anti-DIG antibody-coupled alkaline phosphatase. development. After treatment with 4% PFA for 3 h and extensive washes with PBS, the alkaline In our present study, the temporal and spatial expression profile phosphatase-coupled anti-FITC-antibody was applied in the same way, and the of TeM during chick development was determined and compared staining reaction was performed with INT/BCIP (Roche). Some embryos were embedded in OCT compound (Sakura) following whole mount in situ with the expression domains of Scx and MyoD. Scx expression hybridization from which 20–30 μm sections were obtained. Appropriate control was found to precede TeM expression at the early stages of experiments were performed to exclude false-positive staining due to the development in the musculoskeletal system. At stage 23, Scx endogenous alkaline phosphatase activity. expression in the syndetome extended to the tail region, whereas For in situ hybridization analysis of frozen sections, chick embryos were TeM was only detectable in the anterior eight somites. At stage 28, infiltrated with 18% sucrose/PBS at 4°C, followed by embedding in OCT compound and storage at −80°C. For histological staining and in situ a region-specific expression profile of TeM was observed along hybridization on paraffin sections from the same specimen, chick embryos the craniocaudal axis, depending on the levels of the vertebrae. At were fixed in 4% PFA overnight, dehydrated in a graded series of ethanol and stage 32 and later, either cord- or sheet-like expression domains of embedded in paraffin. Sections were then cut at a 6–10 μm thickness and TeM and Scx were observed in developing tendons. We also mounted onto microscope slides for processing. For RNA probes, the cDNAs detected the expression of TeM in tenocytes isolated from leg for TeM, ChM-I and MyoD were amplified by RT-PCR based on their published sequences in GenBank (NM206985, NM204810 and L34006, respectively). tendons at stage 41. Using an in vitro culture system for tenocytes, The Scx RNA probe was generated from its entire coding sequence in GenBank we found that the retroviral expression of Scx results in the (AF505881), and from pcScx3′ UTR, which was a generous gift from Dr. Tabin. upregulation of TeM in tenocytes. This upregulation was not Type I collagen alpha 2 (Col1a2) and type II collagen alpha 1 (Col2a1) observed in cultured chondrocytes that were overexpressing Scx. constructs were generously provided by Dr. BR. Olsen and used as templates for Consistent with these results, in ovo electroporation of RCAS-Scx the generation of their respective RNA probes. into hindlimb did not induced tendon expansion nor the Immunohistochemistry production of additional tendons, but resulted in the upregulation of TeM in the tendons at stage 33 and later. These data clearly Following in situ hybridization, embryo sections were incubated with suggest that TeM is a good phenotypic marker for tenocytes, and primary antibody at 4°C overnight, washed several times in PBS and 236 C. Shukunami et al. / Developmental Biology 298 (2006) 234–247 reincubated with biotinylated horse anti-mouse secondary antibody (Vector constructs using FuGENE6 (Roche), and the transfected cells were then Laboratories Inc.) for 30 min. Myosin heavy chain was detected with MF20 propagated. After two passages, the medium was replaced with fresh MEM (diluted 1:5000; Developmental Studies Hybridoma Bank). Type II collagen supplemented with 1% FCS. After 24 h, the medium was harvested and was detected with Mouse anti-collagen Type II (diluted 1:200; RDI). concentrated by Centri-Sep (Applied Biosystems). Concentrated retroviral

Endogenous peroxidase activity was inactivated by incubation in 0.3% H2O2/ preparations were then used for the transfection of tenocytes and chondrocytes. PBS for 30 min, following the addition of secondary antibody. Sections were washed and incubated with ABComplex, and immunoreactivity was detected In ovo electroporation using the substrate kit for peroxidase of DAB or Nova RED (Vector Laboratories Inc.). Fertilized eggs were purchased from the Takeuchi poultry farm and incubated until the chick embryos reached Hamburger–Hamilton stages 16–17. Histological staining A small window was opened for access and PBS was poured over the embryo to obtain an appropriate level of resistance (<0.5 Ω). A CUY-21 electroporator For azan staining, deparaffinized and hydrated sections were immersed in an (Gene System) was used according to a previously reported method (Kida et al., azocarmine G solution for 30 min at 56–60°C, rinsed with an aniline alcohol 2004; Takeuchi et al., 1999). Briefly, two platinum electrodes were used as an solution and 1% acetic acid in ethanol, soaked in 5.0% phosphotungstic acid anode and a cathode. The anode was inserted beneath the embryonic endoderm aqueous solution for 60 min at room temperature and dipped in aniline blue- and the cathode was placed on the ectoderm surface of the right hindlimb bud. A μ μ μ μ orange G solution for 30 min at room temperature. DNA solution (5 g/ l) was added to the pCX-EGFP vector (5 g/ l) at a 2:1 ratio and then injected by a sharp glass pipette in the lateral plate mesoderm under the right hindlimb bud. Electric pulses were then applied (9 V, 30 ms Cell culture pulse-on, 70 ms pulse-off, three times) during the injection of the DNA. Following trypsin and collagenase digestion, tenocytes were isolated from leg tendons of chick embryos at stages 41 or 43 and from distal tibiotarsal cartilage chick embryos at stage 41, respectively. Isolated tenocytes and Results chondrocytes were grown in αMEM medium containing 10% FBS and DME/ F12 medium containing 10% FBS, respectively, in a humidified atmosphere of Cloning and sequence analysis of the chick TeM cDNA 5% CO2 in air. TeM was cloned as a related gene of ChM-I, which was Western blot analysis previously purified from bovine epiphyseal cartilage based on Samples were electrophoresed on 12.5% SDS-polyacrylamide gels and its growth stimulatory activity in chondrocytes (Hiraki et al., transferred to Immobilon-P membranes (Millipore). After preincubation with 1991), and later identified as a vascular endothelial blocking buffer (5% nonfat milk, 0.1% Tween 20 and 0.1% NaN3 in PBS), the inhibitor (Brandau et al., 2001; Hiraki et al., 1997; Shukunami membranes were incubated with anti-FLAG monoclonal antibody M2 (Sigma- et al., 2001; Yamana et al., 2001). TeM is a type II trans- Aldrich), followed by horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody and HRP-conjugated anti-mouse IgG antibody (Amersham Pharmacia membrane protein with an anti-angiogenic domain at its C- Biotech), respectively. Peroxidase activity was detected and visualized using the terminus (Oshima et al., 2003, 2004). To study the role of TeM enhanced chemiluminescence (ECL) plus system (Amersham Pharmacia in the chicken embryo, we isolated the chicken homolog of Biotech), according to the manufacturer's instructions. mouse TeM and found that it contains an open reading frame of 957 nucleotides encoding 319 amino acids (Fig. 1A). A Kyte– Northern blot analysis Doolittle hydrophilicity plot further suggested that chick TeM is also a type II transmembrane protein with a short cytoplasmic Total RNA was prepared from cultured tendon fibroblasts, chondrocytes and from tendons isolated at HH stage 41 by a single step method. Total RNA tail, followed by a hydrophobic transmembrane region with (15 μg) was denatured with 6% formaldehyde, fractionated by 1% agarose gel its C-terminus expressed on the exterior cell surface (Fig. 1B). electrophoresis and transferred onto Nytran membranes with Turboblotter Two possible N-glycosylation sites in the BRICHOS domain (Schleicher and Schuell). Hybridization was performed overnight at 42°C with were found at positions 95 and 181, both of which are α 32 an appropriate cDNA probe labeled with [ - P]dCTP from Amersham conserved in mammals (Fig. 1A) (Sanchez-Pulido et al., Biosciences in a solution containing 50% formamide, 6× SSPE, 0.1% bovine serum albumin, 0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.5% SDS and 2002). In addition, the chicken sequence has 62% overall 100 μg/ml denatured salmon sperm DNA. identity with that of mouse or human. However, the ChM-I like domain from Phe257 to Val319 is a highly conserved moiety, Plasmid construction which includes eight Cys residues, across the different species. Within this domain, the sequence is identical The coding region of chick Scx was amplified by PCR with the following between mouse, rat, human and horse, and the homology was primers: forward (5′-GCTCGCCATGTCCTTCGCCATGC-3′) and reverse (5′- 87% between chick and mammals. The lowest level of ′ TTCCCCGGGAGCGCCTAGCTCC-3 ). The amplified products were sub- similarity among the mammalian TeM was observed cloned into the p3xFLAG-CMVTM-10 expression vector to generate Flag- tagged Scx. Flag-tagged Scx or untagged Scx inserts were then ligated to a from Ala198 to Lys234, between the BRICHOS domain and the pSLAX vector and subcloned into an RCASBP(A) retrovirus vector. The coding ChM-I like domain. region of EGFP was subcloned into RCASBP(A) or RCASBP(B) retrovirus The genomic sequence of the chick TeM gene is available vector. The coding region of chick was amplified by PCR with the from the chicken Ensemble project. Chick TeM has been ′ following primers: forward (5 -CCATCGATAGCCGTGCACAGTCCTCCC- localized to chromosome 4. The sizes and boundaries of the 3′) and reverse (5′-CCATCGATCCAGCTCAGTTTTGGACCCG-3′). The amplified products were subcloned into the Cla-1 site of RCASBP(A) retrovirus exons and introns have been analyzed. As shown in Fig. 1C, the vector. RCASA-mPax1 is a generous gift from Dr. K. Imai (Rodrigo et al., 2003). chick TeM gene spans approximately 7.76 kb and consists of Chicken embryonic fibroblasts (CEF) were transfected with these retrovirus seven exons, which all follow the AG-GT splicing consensus C. Shukunami et al. / Developmental Biology 298 (2006) 234–247 237

Fig. 1. A comparison of the deduced amino acid sequences of TeM proteins, Kyte–Doolittle Hydrophilicity plots and genomic organization of chick TeM. (A) Predicted amino acid sequences of chick, mouse (Brandau et al., 2001; Shukunami et al., 2001; Yamana et al., 2001), human (Shukunami et al., 2001) and horse TeM. The chicken sequence shares 62% homology with the previously cloned mouse, rat and human orthologs and 63% overall identity with horse. The conserved amino acid residues are shaded and gaps were introduced for optimal alignment. The putative transmembrane domains are underlined and conserved cysteine residues are indicated in bold. The potential N-linked glycosylation sites are denoted by asterisks. GenBank accession numbers for the aligned sequences are as follows: chick TeM, AY156693; mouse TeM, AF219993; rat TeM, NM02290; human TeM, AF234259; horse TeM, AB059407. (B) Kyte–Doolittle hydrophilicity plot of chick TeM. (C) The chick TeM gene has seven exons and spans 7.76 kb. Cytoplasmic portion and transmembrane domain are encoded by the first two exons. The extracellular domain is encoded by exon2–7.

(not shown). Exon1 and exon2 encode the cytoplasmic N- Induction of TeM is associated with the formation of tendon terminal region and the transmembrane region, respectively. primodia during chick development The extracellular region of the protein is encoded from exon2 to exon7. Exon7 contains the complete C-terminal anti-angiogenic Scx is a marker of the syndetome, a sclerotomal subdomain domain, incorporating the eight Cys residues. containing a tendon progenitor population (Brent et al., 2003). 238 C. Shukunami et al. / Developmental Biology 298 (2006) 234–247

Scx expressing cells are present at the anterior and posterior syndetome (Fig. 2K). Further analysis using transverse sections somitic borders along the cranio-caudal axis at stage 23 (Fig. of hybridized embryos revealed that the TeM and Scx double 2A). In contrast, TeM expression is detectable only in the positive cell populations are flanked by a single TeM positive anterior eight somites at stage 23 (Fig. 2B). In the cranial four cell population and Scx positive cell population (Figs. 2L, M). somites, the faint expression of TeM was evident, but higher By dissecting the hybridized embryos from the back in a frontal expression levels of TeM were visible in the posterior border of direction, we also confirmed that TeM single positive cell the fifth and in the anterior border of the sixth somite, populations (purple) are present in the dorsomedial region (Fig. followed by weaker signals in the seventh and eighth somite 2N), followed by TeM/Scx double positive cell populations (Fig. 2B). In the developing forelimb and hindlimb at stage 23, (Fig. 2O). A Scx single positive cell population (orange) was Scx can be detected in the proximomedial domains (Fig. 2A), then found to be present in the ventrolateral region (Fig. 2P). whereas TeM was found to be diffusely expressed in the posterior region (Fig. 2B). TeM is expressed in developing chick tendons The cranial four somites contribute to the formation of occipital bone and the remaining somites give rise to the At stage 30, TeM positive domains were segmentally vertebrae (Christ et al., 2000). The chick vertebral system arranged along the vertebral column (Fig. 3A). Scx was comprises 14 cervical vertebrae (C1–C14), 7 thoracic vertebrae similarly expressed, but also detectable in the intercostal (T1–T7), 4 lumber vertebrae, 9 sacral vertebrae (L1–L13) and regions (Fig. 3B). Both TeM and Scx were detectable in the ∼13 coccyges. At stage 28, more divergent expression patterns regions between MyoD positive domains that were also for TeM and Scx were observed, probably reflecting the region- segmentally arranged at this stage (Fig. 3C). However, TeM specific development of muscles and skeletal elements along positive domains started to fuse at stage 31 (Fig. 3D) and the developing vertebral column (Figs. 2C–E). At the upper became cord- or sheet-like at stage 32 (Fig. 3E). TeM marks region of the cervical vertebrae, the fusion of Scx and TeM both the developing axial tendons and the attachment sites of expressing domains in the anterior and posterior borders of the tendons to the skeletal elements at stage 32 (Figs. 3E, H). somites could be observed (Figs. 2C, D), and intervals between Strong signals were also observed in the dense connective each myotome that expressed MyoD were found to have tissues surrounding the pelvis (Fig. 3E). In addition, a TeM narrowed (Fig. 2E). From the fifth to ninth somite, TeM was positive domain was detected in the region along the scapula detectable at a higher level (Fig. 2D). At the thoracic and lumber (arrowheads, Fig. 3E). At the thoracic level, however, TeM was levels, Scx and TeM were expressed in the anterior and posterior only detectable in the proximal regions of the ribs at this stage borders of the sclerotome, beneath the myotome, in a double (arrows, Fig. 3E). Scx was also observed to be a marker for the stripe pattern (Figs. 2C, D). The ventrolateral expression of Scx developing chick tendons, but its expression was found to be was extended to the regions adjacent to the pectoral and more associated with the muscular components (Figs. 3F, G). abdominal muscles (arrows, Fig. 2C), whereas TeM was only The positive expression of Scx was clearly evident in the found to be expressed in the dorsomedial region at the thoracic surrounding regions of the intercostal muscles (black arrows, region (asterisk, Fig. 2D). It should be noted here that the Figs. 3F, G), the lateral iliotibial muscle (asterisk, Figs. 3F, G) dorsomedial–ventrolateral expression of TeM was observed at and in the tendons connecting the scapula with the rhomboid the cervical and lumber levels (Fig. 2D). In the dorsal view, a and scapulohumeral muscle (red arrowheads, Fig. 3F). similar v-shaped pattern of expression for TeM and Scx was At stage 35, characteristic cord- or sheet-like structures of observed and these patterns persisted along the axial skeleton tendons can be visualized as expression domains of TeM (Fig. until stage 31 (not shown). At the limb level, the expression of 4A). Similarly, TeM and Scx are markers of the developing TeM was observed in the shoulder and pelvic girdles, and the tendons in the wing at this stage (Figs. 4B, C). TeM was also proximal region of the wing and leg (Fig. 2D). In the saggital found to be expressed in nontendinous dense connective tissues, sections at the thoracic level, overlapping expression of Scx and such as dermis, at stage 36 (arrowheads, Fig. 4D), whereas Scx TeM was detected in the dorsal region (Figs. 2F, G). Brent et al. expression was not detectable at this point (Fig. 4E). During previously reported that Scx positive domain is detectable just stage 36, tenocytes synthesize type I collagen fibrils, but mature adjacent to the myotome at stage 20 (Brent et al., 2003). fibers of type I collagen, which stain dark blue via the azan Similarly, at stage 28, MyoD positive domain was detected method, were not observed (not shown). We then compared the between Scx or TeM positive domains (Figs. 2F–H). expression domain of TeM with that of Scx in immature To determine the precise expression domains of TeM during tendons. As shown in Figs. 4F and G, TeM is expressed in dense axial development, we performed two-color whole mount in connective tissues located between the vertebrae, whereas Scx situ hybridization analysis using stage 25 chick embryos. MyoD is not detectable in the corresponding regions. Scx expression expression was observed to span the full dorsomedial– was not detected in the intervertebral region at stages 29 and 41 ventrolateral length of the somites (Fig. 2I). Moreover, TeM (not shown). In contrast, in the trunk, Scx was clearly evident in and MyoD act as markers for two adjacent but not overlapping the myotendinous junction of the intercostal muscles (Fig. 4H), cell populations at this developmental stage, as shown by a where TeM was undetectable at this stage (Fig. 4I). At stage 41, lateral view (Fig. 2I) and by a frontal section (Fig. 2J). In mature fibers of dense connective tissues are formed in chick contrast, overlapping expression domains were observed for (Fig. 4K) and TeM can be detected in both the myotendinous TeM and Scx, indicating that TeM is also expressed in the and osteotendinous junctions during this time (Fig. 4J). Thus, C. Shukunami et al. / Developmental Biology 298 (2006) 234–247 239

Fig. 2. Comparative analysis of TeM and Scx gene expression during chick development. (A) Scx is detectable in the anterior and posterior somitic borders along the cranio-caudal axis and the proximomedial domain of the wing bud (arrowhead) at stage 23. (B) TeM is detectable in the posterior border of the fifth somite (arrow), the anterior and posterior edges of the following three somites and the posterior domain of the wing bud (arrowhead) at stage 23. (C) At stage 28, Scx is expressed in a double stripe pattern between the somites, dorsomedially and ventrolaterally along the cranio-caudal axis, except for the upper cervical region in which fusion of the anterior and posterior borders of each somite occurs. In the thoracic region, Scx is detected in the ventrolateral domain, which coincides with the incipient ribs and intercostal muscles (red arrows). (D) TeM is expressed dorsomedially along the edges of the somites and the connective tissues of the shoulder joint, pelvic region, jaw and limbs at stage 28. At the cervical level, strong dorsomedial expression is observed from the posterior border of the fifth somite to the anterior border of the ninth somite. Weaker signals are detected in the following seven somites. In the ventrolateral region, TeM is similarly expressed from the fifth to the nineteenth somite. Note that ventrolateral expression of TeM is not detectable in the thoracic region (asterisk). (E) Full dorsomedial–ventrolateral expression of MyoD. In the cervical region, the intervals between each myotome expressing MyoD become narrower. (F–H) In situ hybridization of Scx, TeM and MyoD in the saggital semiserial sections prepared from india ink injected embryos at stage 28. Ventral is towards the left and dorsal is towards the right. Scx expression in the syndetome is shown (F). Overlapping expression of TeM with Scx is detected in the dorsal region (F, G). MyoD is expressed in the myotome between Scx positive regions (H). (I) Two color whole mount in situ hybridization analysis of TeM and MyoD at stage 25. TeM (purple) and MyoD (orange) mark two adjacent but nonoverlapping cell populations, shown dorsolaterally. (J) The frontal section of a chick embryo detected with TeM (purple) and MyoD (orange) shows nonoverlapping expression. (K) Two color in situ hybridization analysis of TeM and Scx shows overlapping expression in the anterior and posterior edges of the cervical somites at stage 25. (L, M) Embryos detected with TeM and Scx by two color in situ hybridization were embedded, and transverse and frontal sections were prepared from the cervical region. The inset in L corresponds to the area in M. In the transverse section, double positive expression domains of TeM and Scx are flanked by a dorsomedial TeM single positive domain and a ventrolateral Scx single positive domain (M). Frontal sections from the dorsal to ventral direction are shown in panels N–P. Following the dorsal TeM single positive region (N), TeM and Scx double positive regions appear (O) followed by a Scx single positive region (P). The thickness of the sections (N, O, P) is 30 μm. The intervals thickness among the section from N to O and from O to P is 30 and 150 μm, respectively. Scale bars, 100 μm. our analysis reveals the predominant expression of Scx in the TeM was originally cloned as a ChM-I-related gene that was myotendinous junction and of TeM in the chondrotendinous found to be predominantly expressed in the avascular cartilage junction in immature tendons. (Brandau et al., 2001; Shukunami et al., 2001; Yamana et al., 240 C. Shukunami et al. / Developmental Biology 298 (2006) 234–247

Fig. 3. Distinctive expression patterns of TeM and Scx during development of the musculoskeletal system. At stage 30, TeM and Scx are segmentally expressed in the regions between MyoD positive domains (A–C). TeM is detectable in the center of backbone (A, arrowheads). Scx is detectable at the ventrolateral domain coincides with the incipient ribs and intercostals muscles (B, asterisk). At stage 31, adjacent TeM positive domains begin to fuse (D). (E–H) Skeletal preparations of the thoracolumbar region of a chick embryo at stage 32 are shown in comparison with dorsolateral views of stage 32 embryos hybridized with TeM, MyoD and Scx riboprobes. Postulated regions of the scapula and pelvic girdle are depicted by a dotted line. TeM transcripts are detected in tendons and ligaments connecting the pelvic girdle with the surrounding muscle masses and bone rudiments, and the attachment sites of the connective tissues to the scapula (arrowheads) and the proximal region of ribs (arrows) (F). Scx is predominantly detected in the connective tissues on the outer margins of the muscle masses (arrows and asterisk in panel F) and in the tendons connecting the scapula with the surrounding muscle masses (arrowheads in panel F). Intercostal, leg and wing muscles are denoted by MyoD (G). shm, scapulohumeral muscle; icm, intercostal muscle; lim, lateral iliotibial muscle (M. iliotibialis lateralis); itm, iliotrochanteric msucle; rb, rib; s, scapula; f, femur; pl, pelvic girdle; T1, thoracic vertebra 1; L1, Lumber vertebra 1.

2001). TeM was also subsequently identified as one of a group tissues (Figs. 5C, D, F). Maturation of tendons is associated of downregulated genes in a muscle atrophy model (Pisani et with formation of the laminar configuration where oval and al., 2004). To examine the precise expression domains of TeM elongated tenocytes appear (Chuen et al., 2004). Immature oval in the developed musculoskeletal system in the chick, we tenocytes derived from tendon progenitors rapidly proliferate performed a combination of in situ hybridization and immu- and then mature to become elongated tenocytes. At a higher nostaining analysis. As shown in Fig. 4L, TeM was observed to magnification, elongated tenocytes are aligned in parallel to be expressed in tendons that were located along the dorsal thick mature collagen fibers to form the regular layer, whereas muscles and that were connecting muscle with the spinous oval tenocytes are randomly distributed in the interlaminar processes at the cervical level. In the shoulder region, TeM was spaces (Figs. 5G–I). Although Col1a2 expression was detected also shown to be expressed in tendons binding either the scapula in both oval and elongated tenocytes (Figs. 5G, K), TeM or the vertebral arches with adjacent muscle masses, but not in expression was restricted to elongated tenocytes (Figs. 5H, J). muscle fibers (Fig. 4M). TeM is also specifically expressed in These data suggest that TeM is a marker of mature tenocytes in the heel flexor and extensor tendons and is undetectable in vivo. hyaline cartilage that positively stains with anti-type II collagen antibodies (Fig. 4N). In addition, TeM cannot be detected in TeM is a marker for tenocytes in vitro muscle fibers but is evident in the myoseptum of the leg skeletal muscle, which can be positively stained with anti-myosin heavy Fibroblasts in tendons and ligaments in vivo are histologi- chain antibodies (Fig. 4O). cally distinct and can be characterized by longitudinal rows of Azan staining of heel sections revealed that dense type I winged cells, separated by an extracellular matrix. However, collagen fibers were distributed in tendons, dermis and cortex these cells are almost indistinguishable from fibroblasts in loose bone at stage 41 (Fig. 5A). TeM was only detectable, however, connective tissues in vitro, since their histological character- in the dense connective tissues where thick bundles of collagen istics are lost in these conditions. To examine whether TeM is fibers can be found (Figs. 5B, D, E). In contrast, expression of expressed in vitro, we isolated tenocytes from leg tendons at Col1a2 was detectable in both dense and loose connective stage 41 chick embryos using both trypsin and collagenase C. Shukunami et al. / Developmental Biology 298 (2006) 234–247 241

Fig. 4. Expression of TeM in musculoskeletal tissues. (A) At stage 35, TeM is expressed in appendicular and axial dense connective tissues. (B–C) Similar but distinctive expression patterns of TeM (B) and Scx (C) are observed in the wing at stage 35 as a whole mount. (D–I) In situ hybridization of TeM or Scx, followed by immunostaining, was performed on sections through embryos at stage 36. Muscle was detected with myosin heavy chain antibody (red brown), and cartilage with anti- type II collagen antibody (yellow brown). In wing sections, TeM is detectable in the attachment sites of the tendon to the proximal end of the ulna (arrow in panel D) and the dermis (arrowheads in panel D), whereas Scx is undetectable in these regions (E). In the frontal sections of the vertebral column, TeM is evident in the intervertebral region (arrowheads in panel F), but Scx is not (G). In the thoracic level of the trunk, Scx is expressed in the connective tissue surrounding the intercostal muscles (arrows in H), whereas TeM is undetectable here at this stage (I). (J–K) At stage 41, in situ hybridization analysis of TeM (J) and Azan staining of collagen fibers (K) is performed on serial paraffin sections of the trunk. TeM is detectable in the region surrounding the intercostal muscles (arrows in panel J), associated with the dense fiber distribution observed as dark blue Azan staining (arrowheads in K). (L–O) In situ hybridization of TeM (purple) followed by immunostaining was performed on embryo sections at stage 35. Muscle was detected with myosin heavy chain antibody (red brown in panels L, M and O), and cartilage with anti-type II collagen antibody (yellow brown in panels L and M, or brown in N). In the frontal section of the backbone tissue, TeM is expressed in tendons along the dorsal muscles and in tendons connecting muscle with the spinous processes of the cervical vertebrae (L). In the shoulder region, TeM is detectable in the tendons connecting the scapula and vertebral arches with adjacent muscle masses (M). In the sagittal section of the leg, TeM transcripts are present in the developing heel flexor and extensor tendons (N). At higher magnification, TeM transcripts are evident in connective tissue encompassing muscle masses (O). u, ulna; fb, feather bud; vb, vertebral body; nc, notochord; rb, rib; icm, intercostals muscle; sp, spinous process; va, vertebral arch; s, scapula; ti, tibiotarsus. Scale bars, 100 μm. digestion methods. The isolated tenocytes exhibited a fibro- both tenocytes and chondrocytes (Fig. 6C). Thus, TeM could be blastic morphology (Fig. 6A) and were found to express TeM, a promising marker for tenocytes in the monolayer culture. Col1a2, ChM-I, Col2a1 and Scx (Fig. 6C), although the mRNA levels for TeM in these in vitro experiments are lower than the Scx positively regulates the expression of TeM in tenocytes levels in tendons. We also isolated chondrocytes from the distal tibia with trypsin and collagenase, and these cells exhibited a To explore the functional roles of Scx in the regulation of polygonal morphology (Fig. 6B) and expressed ChM-I, Col2a1 tendon and cartilage cells, we overexpressed Scx in cultured and Aggrecan (Fig. 6C, not shown). The expression of TeM was tenocytes and chondrocytes using retroviruses. When expressed not detectable in chondrocytes, whereas ChM-I was not evident in chick embryonic fibroblasts, Flag tagged Scx displays a in either tenocytes or in tendons (Fig. 6C). Scx was detected in single band of ∼28 kDa, the expected size (Fig. 7A) (Cserjesi et 242 C. Shukunami et al. / Developmental Biology 298 (2006) 234–247

Fig. 5. Expression of TeM in elongated tenocytes of leg tendons. (A) Azan staining of a sagittal section of leg at stage 41. Dense collagen fibers consisting of type I collagen in tendons (t), cortex bone (cb) and periosteum (po) as dark blue dense bundles. Thin type II collagen fibrils in cartilage (ca) stain light blue, whereas nuclei from epidermis (epi) stain red. Blue collagen fibers are sparsely present in loose connective tissues (lc) between tendons and relatively thick blue collagen fibers are also present in dermis (de). (B) In a semiserial section, TeM is detectable in dense connective tissues where dark blue bundles as shown in Azan staining are present. (C) Col1a2 is detected in both dense connective tissues and loose connective tissues. (D–F) The insets in panels A, B and C correspond to the area shown in panels D, E and F, respectively. TeM expressing domain is restricted to the dense connective tissues, showing laminar distribution running parallel to the longitudinal axis of the tendon (D, E). Col1a2 is expressed in both dense connective tissues and loose connective tissues (D, F). (G–I) A semiserial section stained with hematoxylin and eosin is shown in panels G, H and I. The equivalent semiserial region shown in the inset in panel D corresponds to the area shown in panel G. Tendons consist of layer of oval tenocytes (LOT, black arrow) and layer of elongated tenocytes (LET, blue arrow). At higher magnification, mature tenocytes exhibiting an elongated morphology in LET (H) and immature tenocytes exhibiting an oval morphology (I) are shown. (J, K) The insets in panels E and F are corresponding to the area shown in J and K, respectively. Nomarski differential interference contrast images are shown at higher magnification. Expression of TeM is predominantly detected in elongated or flattened nuclei of mature tenocytes (J), whereas Col1a2 is intensely expressed in both oval and elongated tenocytes (K). me, metatarsal bone; ti, tibia. Scale bars, A– C=1 mm, D–F=100 μm and G–K=10 μm. al., 1995). The morphology of the tenocytes overexpressing Scx electroporated retroviral vectors directly into the hindlimbs of was similar to tenocytes overexpressing EGFP (Figs. 7B–D). stage 16–17 embryos. Unlike the injection of infectious Although Scx overexpressing chondrocytes also exhibited a retrovirus particles, electroporated retroviral DNA gradually similar morphology to chondrocytes expressing EGFP (Figs. propagates in the hindlimb and env expression was detectable in 7E–G), slight increases in alcian blue positive extracellular the right leg of stage 28 embryos by whole mount in situ matrices were observed in day 6 chondrocyte cultures infected hybridization (Fig. 8A). Since obvious morphological pheno- with RCAS-cScx (not shown). types were not evident, we analyzed the expression of TeM in We next examined the mRNA levels of various marker stage 30, 33 and 35 embryos by whole mount in situ genes, including TeM and ChM-I, by Northern blotting. TeM hybridization. We did not detect any significant changes in was significantly upregulated in Scx overexpressing tenocytes the right leg, that was misexpressing Scx, until stage 30 (Fig. but was downregulated together with TN by overexpression 8B). At stage 33, the propagation of RCAS-Scx throughout the of Pax-1, a sclerotome marker that inhibits Scx expression in leg was observed (Fig. 8C) and then the significant upregulation the syndetome (Fig. 7H) (Brent and Tabin, 2004). In of TeM was detectable in the right leg tendons of 7/20 embryos, chondrocytes, Scx fails to induce TeM, but a slight when compared with the contralateral left leg (Fig. 8D). At upregulation of Agg and ChM-I could be detected (Fig. stage 35, the upregulation of TeM was detectable in 12/20 7H). We then misexpressed Myogenin as other type of b-HLH embryos (Fig. 8E). The effect of Scx overexpression was not type in tenocytes. Northern blot analysis site-specific, consistent with our in vitro data that tenocytes revealed that TeM and Scx expression was significantly isolated from various leg tendons were equally responsive to downregulated in tenocytes that misexpressed Myogenin to Scx. In situ hybridization analysis revealed that TeM expression form myotoubes in the presence of 0.5% FBS (Fig. 7I). This in the infected or the contralateral legs was only evident in is consistent with our observation that TeM was not expressed developing tendons and other dense connective tissues (Figs. in myogenic or muscle cells (Figs. 2, 3, 4O). In addition, the 8F, I), even though the retroviral expression of Scx was data suggested that TeM upregulation by Scx was not simply observable in the entire leg, including muscle and loose attributed to a genial b-HLH activity, but specific to Scx. connective tissue (not shown). Consistent with our in vitro We also tested Scx function in the developing hindlimb, analyses, the expression of TeM was not found outside the taking advantage of in ovo electroporation technology. We endogenous expression domains (Figs. 8F–I). Upregulation of C. Shukunami et al. / Developmental Biology 298 (2006) 234–247 243

Indeed, at stage 28, the expression of TeM becomes more regionally characteristic than Scx (Figs. 2C, D). As chick vertebrae display distinct morphological features at different levels, TeM was also expressed differently in the cervical, thoracic and lumbosarcal regions. In the appendicular region, tendon primordia divide into individual tendons that interact with developing muscles and skeletal elements (Kardon, 1998). In the axial region, each tendon primordium should be reorganized to form individual tendons due to the polymerization of epaxial muscles (Christ et al., 2000). As the superficial part of the segmentally arranged epaxial muscle anlagen fuse to give rise to segment-over- bridging muscles, already existing tendons lose their connection with the muscle and disappear. Monitoring TeM and Scx expression, we found that the positive domains of these genes became cord- or sheet-like at around stage 32. At the initial stages of tendon development, the expression of TeM and Scx does not completely overlap. Indeed, simultaneous detection of Scx and TeM by two color in situ hybridization of the cervical regions in a stage 25 embryo reveals three different populations along the dorsoventral axis (Fig. 2M). TeM single positive cell populations are observed dorsally adjacent to TeM/ Scx double positive cells (Figs. 2M–O). In the ventral region of TeM/Scx double positive cells, Scx single positive cell populations are present (Figs. 2M, O, P). Grafting experiments using chick–quail chimera showed that axial tendon cells are of sclerotomal origin (Brent et al., 2003). However, recent analysis of Ets transcription factors that restrict Scx expression in the somites revealed that Scx was also expressed in small subsets of dermomyotome (Brent and Tabin, 2004). TeM single positive Fig. 6. Expression of TeM in cultured tenocytes. (A) Tenocytes isolated from leg populations in the dorsal region may indicate an additional tendons at stage 41 were grown at a density of 1×105 cells/well in a 6-well plate contribution to tendon formation by dermomyotome cells. for 3 days in αMEM containing 10% FBS. The cells show an elongated However, we could not exclude the possibility that cells fibroblastic morphology. Scale bar, 100 μm. (B) Chondrocytes isolated from previously expressing Scx in the syndetome migrate to the 5 distal tibia at stage 41 were seeded at a density of 3×10 cells/well in a 6-well dorsal region and downregulate Scx due to the influence of plate and were grown for 8 days in DF medium containing 10% FBS. The cells show a polygonal morphology. Scale bar, 100 μm. (C) Northern blot analysis of surrounding tissue, thus allowing cells to express only TeM. TeM, Col1a2, ChM-I, Col2a1 and Scx in chicken tenocytes, chondrocytes and Alternatively, TeM single positive cell populations may tendon tissue. Cells were grown in 6-well plates. Total RNA was isolated from represent the prospective dermis arising from dermomyotome, tenocytes on day 5, from chondrocytes on day 6 and from tendon tissue in day- since TeM is also expressed in nontendinous tissues such as μ 20 chick embryos. RNA (15 g) was loaded in each lane and hybridized with the dermis and ligaments (Figs. 4D, F). respective cDNA probes. Equal loading was verified with ethidium bromide. The positions of 27S and 18S ribosomal RNAs are indicated. Tenocytes lie in longitudinal rows and mature to become very elongated, being 80–300 μm in diameter, and have long and thin cellular processes by which close contact between TeM upon Scx infection was also confirmed by Northern blot adjacent cells and extracellular matrices is maintained analysis (Fig. 8J). (McNeilly et al., 1996). Due to their unique morphological features, tenocytes are easily distinct from other fibroblasts in Discussion loose connective tissues, such as hypodermis and submucosal tissue, in vivo. However, once these cells are isolated from During chick axial tendon development, Scx is first induced tissues and cultured in vitro, their cellular morphology and at stage 18 and its expression intensifies during somite longitudinal alignments are lost and they become indistinguish- maturation (Brent et al., 2003). Moreover, up until stage 23, able from fibroblasts derived from loose connective tissues. The Scx expression in the syndetome extends to the tail region current lack of a phenotypic marker for tenocytes in vitro also (Fig. 2A), whereas TeM is only expressed in the anterior eight makes it difficult to establish a culture system for their induction somites (Fig. 2B). Since segmentation occurs from the cranial to from stem cells. Scx is a good marker of the tendon cell lineage the tail region along the anteroposteior axis, the later and more in vivo, but seems inappropriate to use in vitro due to its restricted expression profile of TeM may reflect the ongoing expression in skeletal lineage cells. Scx expression is detectable formation of tendon primordia at the upper cervical region. in various cell lines derived from the skeletal lineage, such as 244 C. Shukunami et al. / Developmental Biology 298 (2006) 234–247

Fig. 7. Upregulation of TeM in tenocytes infected with RCAS-cScx. (A) Chick embryonic fibroblasts isolated from embryos at stage 41 were infected with RCAS- 3xFLAG-Scx. A single 30 kDa band was detected by Western blot analysis. (B–D) Tenocytes isolated from leg tendons at stage 41 were seeded at 1.5×105 cells/well of a 6-well plate in αMEM containing 10% FBS. On day 2, cells were infected with RCAS-EGFP (B and C) or RCAS-cScx (D) and cultured for another 2 days. (E–G) Chondrocytes isolated from distal tibia were seeded at 3.3×105 cells/well in DF medium containing 10% FBS. On day 2, the cells were infected with RCAS-EGFP (E and F) or RCAS-cScx (G) and cultured for another 5 days. (H) Secondary cultures of tenocytes and primary cultures of chondrocytes were infected with RCAS-EGFP, RCAS-cScx or RCAS-mPax1. Expression of TeM, ChM-I, Aggrecan, Col1a2, Col2a1, Tenascin (TN) and env mRNAs was examined by Northern blotting. Upregulation of TeM was detected in tenocytes overexpressing Scx. Some upregulation of Aggrecan and ChM-I was observed. Equal loading was verified with ethidium bromide. (I) Secondary culture of tenocytes was transfected with RCAS-EGFP or RCAS-cMyogenin. After two passages, expression of TeM and Scx mRNAs was examined by Northern blotting. Downregulation of TeM and Scx was observed in tenocytes misexpressing Myogenin. Equal loading was verified with ethidium bromide.

MC3T3-E1, ROS17/2.8 and ATDC5 (Liu et al., 1997) (un- cular mechanisms underlying tenocyte differentiation and ten- published data), and cultured chondrocytes (Fig. 6C). In don regeneration. contrast, TeM is not expressed in these skeletal cell lines Dense connective tissues, including tendons and ligaments, (unpublished data), but is only detectable in tenocytes in vitro are characterized by regularly oriented type I collagen fibers (Fig. 6C). The combination of Scx as an early marker and TeM (Benjamin and Ralphs, 1997; Kannus, 2000). Although as a late marker should now enable clarification of the mole- tenocytes in monolayer cultures have the ability to synthesize C. Shukunami et al. / Developmental Biology 298 (2006) 234–247 245

Fig. 8. Upregulation of TeM in tenocytes by overexpression of Scx in ovo.(A–D) RCAS-cScx was electroporated into the right hindlimb bud of chick embryos at day 2.5 and then processed for whole mount in situ hybridization at stages 28, 30, 33 and 35. Expression of env (arrows) at stage 28 is shown in the RCAS-cScx electroporated right leg (A). At stage 30, TeM expression is detectable in the developing tendons of the right leg misexpressing Scx at similar levels to the contralateral left leg (B). At stage 33, Scx is overexpressed in the entire right leg (C) and higher TeM levels (arrowheads) are evident in the right leg overexpressing Scx, compared with the contralateral left limb (D). At stage 35, TeM expression in the Achilles tendon is higher in the right leg infected with RCAS-cScx (arrowheads) than in the contralateral side (E). (F–I) At stage 35, in situ hybridization with antisense probe of TeM (F, I), sense TeM probe (G) or Azan staining (H) was performed in the semiserial sections of the right leg infected with RCAS-cScx. Although the expression of TeM was detectable in the leg tendons and other dense connective tissues, the signal was never detected ectopically in cartilage, muscle or loose connective tissues (F, H). Positive signals are undetectable on the section hybridized with sense TeM riboprobe (G). (I) Endogenous expression of TeM is detectable in leg tendons and other dense connective tissues of the contralateral left leg. me, tarsometatarsus; ti, tibiotarsus. Scale bar, 100 μm. (J) RCAS-cScx was electroporated with RCAS-EGFP into the hindlimb. cScx was subcloned into RCASBP(A), whereas EGFP was subcloned into RCASBP(B). Total RNA was extracted from the EGFP positive region and the equivalent region of the contralateral leg, respectively. Total RNA (15 μg) extracted from the contralateral left leg (lane 1) or the RCAS-cScx/RCAS-EGFP infected right leg (lane 2) was loaded and hybridized with TeM or env cDNA probes. Upregulation of TeM is detectable in the RCAS-cScx/RCAS-EGFP infected leg (lane 2). Equal loading was verified with ethidium bromide. large amounts of type I collagen (Fig. 7H), these cells lose their transcription factors transdifferentiates some fibroblasts into characteristic elongated morphology and their fibrils are myoblasts, which can differentiate into skeletal muscle when randomly distributed (Fig. 6A). Northern blots also reveal that cultured in low mitogen medium (Tapscott et al., 1988). To TeM transcripts in cultured tenocytes are at lower levels than explore the effects of Scx overexpression in nontendinous cells, in tendons (Fig. 6C). In contrast, the mRNA levels for Scx in we expressed RCAS-cScx in chondrocytes isolated from distal vitro are similar to those in vivo (Fig. 6C). Higher levels of TeM tibiae, which slightly upregulated aggrecan mRNA levels (Fig. were detectable only in dense regular collagen fibers, stained by 7H) and alcian blue positive extracellular matrices (not shown). azan (Figs. 5A, B, D, E), suggesting a close correlation between However, the induction of TeM in chondrocytes overexpressing TeM expression and the regular alignment of collagen fibers. Scx was not observed (Fig. 7H). In contrast, the overexpression Moreover, TeM expression domain is highly restricted to of Scx in tenocytes significantly upregulates TeM mRNA, elongated tenocytes in mature tendons (Figs. 5G, H, J). Mice despite the induction of the dominant negative factors Id2 and lacking TeM display uneven and rough fibril surfaces on their Id3 (not shown). Col1a2 and TN expression levels and the tendons (Docheva et al., 2005), further indicating that TeM is cellular morphology of tenocytes were unaffected by the involved in the formation of organized tendon structures, which overexpression of Scx (Figs. 7D, H). Conversely, the over- reciprocally affect TeM levels. expression of Pax1 results in downregulation of TeM and TN A number of basic helix–loop–helix type transcription (Fig. 7H), suggesting that Pax1 downregulates phenotypic factors are involved in cellular differentiation (Jones, 2004). markers in the later stages of tendon development. Although Ectopic expression of the myogenic basic helix–loop–helix both tenocytes and chondrocytes are derived from common 246 C. Shukunami et al. / Developmental Biology 298 (2006) 234–247 progenitors in the sclerotome and lateral plate mesoderm, the Brent, A.E., Tabin, C.J., 2004. FGF acts directly on the somitic tendon positive regulation of TeM expression by Scx is dependent on progenitors through the Ets transcription factors Pea3 and Erm to regulate scleraxis expression. Development 131, 3885–3896. the tendon cell lineage. Brent, A.E., Schweitzer, R., Tabin, C.J., 2003. A somitic compartment of tendon Misexpression of Scx in the hindlimb fails to induce extopic progenitors. Cell 113, 235–248. formation of the tendinous primordia or ectopic tendons (Fig. Canty, E.G., Kadler, K.E., 2005. Procollagen trafficking, processing and 8). These findings are in good agreement with a previous study fibrillogenesis. J. Cell Sci. 118, 1341–1353. showing that the expansion of Scx expressing regions by noggin Canty, E.G., Lu, Y., Meadows, R.S., Shaw, M.K., Holmes, D.F., Kadler, K.E., 2004. Coalignment of plasma membrane channels and protrusions did not induce tendinous tissue formation (Schweitzer et al., (fibripositors) specifies the parallelism of tendon. J. Cell Biol. 165, 2001). Although the basic patterns of tendon primordia are 553–5563. unaffected by broad misexpression of Scx in the developing leg, Christ, B., Huang, R., Wilting, J., 2000. The development of the avian vertebral we found significant upregulation of TeM mRNA in tendons column. Anat. Embryol. (Berl) 202, 179–194. overexpressing Scx at stage 33 and later (Figs. 8D, E). Chuen, F.S., Chuk, C.Y., Ping, W.Y., Nar, W.W., Kim, H.L., Ming, C.K., 2004. Immunohistochemical characterization of cells in adult human patellar Upregulation of TeM expression was detected in whole legs tendons. J. Histochem. Cytochem. 52, 1151–1157. overexpressing Scx by Northern blot analysis (Fig. 8J). In Cserjesi, P., Brown, D., Ligon, K.L., Lyons, G.E., Copeland, N.G., Gilbert, D.J., nontendinous tissues, including muscle and cartilage, Scx fails Jenkins, N.A., Olson, E.N., 1995. Scleraxis: a basic helix–loop–helix to induce TeM expression, consistent with our in vitro data protein that prefigures skeletal formation during mouse embryogenesis. – (Figs. 7H, 8F). The altered responsiveness to Scx overexpres- Development 121, 1099 1110. Docheva, D., Hunziker, E.B., Fassler, R., Brandau, O., 2005. Tenomodulin is sion in mature tendons may suggest that the competence of necessary for tenocyte proliferation and tendon maturation. Mol. Cell. Biol. tenocytes in leg tendons is significantly altered at stage 33. 25, 699–705. Considering later onset of TeM induction than that of Scx along Edom-Vovard, F., Duprez, D., 2004. Signals regulating tendon formation during the course of tendon development, it is obvious that additional chick embryonic development. Dev. Dyn. 229, 449–457. factors are required for further differentiation of Scx expressing Edom-Vovard, F., Schuler, B., Bonnin, M.A., Teillet, M.A., Duprez, D., 2002. Fgf4 positively regulates scleraxis and tenascin expression in chick limb tendon progenitors into TeM expressing tenocytes. In addition, tendons. Dev. Biol. 247, 351–366. TeM expression was not overlapped with Scx expression in Hamburger, V., Hamilton, H.L., 1992. A series of normal stages in the dermis and the intervertebral region, suggesting that the development of the chick embryo. 1951. Dev. Dyn. 195, 231–272. expression of TeM was regulated by other factors independently Hiraki, Y., Tanaka, H., Inoue, H., Kondo, J., Kamizono, A., Suzuki, F., 1991. from Scx. Molecular cloning of a new class of cartilage-specific matrix, chondromo- dulin-I, which stimulates growth of cultured chondrocytes. Biochem. Scx binds the E-box consensus sequence (CANNTG) as a Biophys. Res. Commun. 175, 971–977. heterodimer with E12 (Cserjesi et al., 1995) and these motifs are Hiraki, Y., Inoue, H., Iyama, K., Kamizono, A., Ochiai, M., Shukunami, C., found throughout the chick TeM genes (now shown). However, Iijima, S., Suzuki, F., Kondo, J., 1997. Identification of chondromodulin I it is still uncertain whether TeM is a direct downstream target of as a novel endothelial cell growth inhibitor. Purification and its localization Scx. In addition, the expression of TeM in the Scx negative in the avascular zone of epiphyseal cartilage. J. Biol. Chem. 272, 32419–32426. region suggests that other factors are involved in tendon Jones, S., 2004. An overview of the basic helix–loop–helix proteins. Genome development, independently of Scx. To address these questions, Biol. 5, 226. the cis-acting element driving the specific expression of TeM in Kannus, P., 2000. Structure of the tendon connective tissue. Scand. J. Med. Sci. tenocytes and the trans-acting factors binding these regions Sports 10, 312–320. should be identified. Such studies are currently underway. Kardon, G., 1998. Muscle and tendon morphogenesis in the avian hind limb. Development 125, 4019–4032. Kida, Y., Maeda, Y., Shiraishi, T., Suzuki, T., Ogura, T., 2004. Chick Dach1 Acknowledgments interacts with the Smad complex and Sin3a to control AER formation and limb development along the proximodistal axis. Development 131, We thank Drs. B.R. Olsen, C. Tabin and K. Imai for providing 4179–4187. chick type II collagen, pcScx3′ UTR and RCASA-mPax1, Liu, Y., Watanabe, H., Nifuji, A., Yamada, Y., Olson, E.N., Noda, M., 1997. Overexpression of a single helix–loop–helix-type transcription factor, respectively. We also thank Drs T. Ogura and Y. Kida for advice scleraxis, enhances aggrecan gene expression in osteoblastic osteosarcoma on in ovo electroporation, Mr. T. Matsushita and Ms. K. Kogishi ROS17/2.8 cells. J. Biol. Chem. 272, 29880–29885. for the histological studies, Dr. Y. Nishizaki for many helpful McNeilly, C.M., Banes, A.J., Benjamin, M., Ralphs, J.R., 1996. Tendon cells in discussions and Ms. Y. Kubo for her valuable secretarial help. vivo form a three dimensional network of cell processes linked by gap – This study was partly supported by Grants-in-Aid from the junctions. J. Anat. 189 (Pt. 3), 593 600. Oshima, Y., Shukunami, C., Honda, J., Nishida, K., Tashiro, F., Miyazaki, Ministry of Education, Culture, Sport, Science and Technology J., Hiraki, Y., Tano, Y., 2003. Expression and localization of of Japan, and by the Tanabe Medical Frontier Conference. tenomodulin, a transmembrane type chondromodulin-I-related angiogen- esis inhibitor, in mouse eyes. Invest. Ophthalmol. Visual Sci. 44, 1814–1823. References Oshima, Y., Sato, K., Tashiro, F., Miyazaki, J.I., Nishida, K., Hiraki, Y., Tano, Y., Shukunami, C., 2004. Anti-angiogenic action of the C-terminal domain Benjamin, M., Ralphs, J.R., 1997. Tendons and ligaments—An overview. of tenomodulin that shares homology with chondromodulin-I. J. Cell Sci. Histol. Histopathol. 12, 1135–1144. 2731–2744. Brandau, O., Meindl, A., Fassler, R., Aszodi, A., 2001. A novel gene, tendin, is Pisani, D.F., Pierson, P.M., Massoudi, A., Leclerc, L., Chopard, A., Marini, J.F., strongly expressed in tendons and ligaments and shows high homology with Dechesne, C.A., 2004. Myodulin is a novel potential angiogenic factor in chondromodulin-I. Dev. Dyn. 221, 72–80. skeletal muscle. Exp. Cell Res. 292, 40–50. C. Shukunami et al. / Developmental Biology 298 (2006) 234–247 247

Rodrigo, I., Hill, R.E., Balling, R., Munsterberg, A., Imai, K., 2003. Pax1 and Smith, T.G., Sweetman, D., Patterson, M., Keyse, S.M., Munsterberg, A., 2005. Pax9 activate Bapx1 to induce chondrogenic differentiation in the Feedback interactions between MKP3 and ERK MAP kinase control sclerotome. Development 130, 473–482. scleraxis expression and the specification of rib progenitors in the Ros, M.A., Rivero, F.B., Hinchliffe, J.R., Hurle, J.M., 1995. Immunohistolo- developing chick somite. Development 132, 1305–1314. gical and ultrastructural study of the developing tendons of the avian foot. Takeuchi, J.K., Koshiba-Takeuchi, K., Matsumoto, K., Vogel-Hopker, A., Anat. Embryol. (Berl) 192, 483–496. Naitoh-Matsuo, M., Ogura, K., Takahashi, N., Yasuda, K., Ogura, T., 1999. Sanchez-Pulido, L., Devos, D., Valencia, A., 2002. BRICHOS: a conserved Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds. Nature domain in proteins associated with dementia, respiratory distress and cancer. 398, 810–814. Trends Biochem. Sci. 27, 329–332. Tapscott, S.J., Davis, R.L., Thayer, M.J., Cheng, P.F., Weintraub, H., Lassar, Schweitzer, R., Chyung, J.H., Murtaugh, L.C., Brent, A.E., Rosen, V., Olson, A.B., 1988. MyoD1: a nuclear phosphoprotein requiring a E.N., Lassar, A., Tabin, C.J., 2001. Analysis of the tendon cell fate using homology region to convert fibroblasts to myoblasts. Science 242, Scleraxis, a specific marker for tendons and ligaments. Development 128, 405–411. 3855–3866. Tucker, R.P., Spring, J., Baumgartner, S., Martin, D., Hagios, C., Poss, Shukunami, C., Oshima, Y., Hiraki, Y., 2001. Molecular cloning of P.M., Chiquet-Ehrismann, R., 1994. Novel tenascin variants with a tenomodulin, a novel chondromodulin-I related gene. Biochem. Biophys. distinctive pattern of expression in the avian embryo. Development 120, Res. Commun. 280, 1323–1327. 637–647. Shukunami, C., Oshima, Y., Hiraki, Y., 2005. Chondromodulin-I and Yamana, K., Wada, H., Takahashi, Y., Sato, H., Kasahara, Y., Kiyoki, M., 2001. tenomodulin: a new class of tissue-specific angiogenesis inhibitors found Molecular cloning and characterization of CHM1L, a novel membrane in hypovascular connective tissues. Biochem. Biophys. Res. Commun. 333, molecule similar to chondromodulin-I. Biochem. Biophys. Res. Commun. 299–307. 280, 1101–1106.